Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) remains a major global public health crisis despite being a curable disease and is the leading cause of morbidity and mortality in both HIV infected and immune competent persons. Our inability to eradicate TB stems in part from our poor understanding of how the cell division (CD) and cell wall synthesis (CWS), the basic essential processes critical for Mtb multiplication, are carried out and importantly regulated in response to infection. We recently discovered that two small novel membrane proteins, CrgA and CwsA, that interact with each other and other CD proteins are important for CD and CWS. However, the activities of these proteins and the biological consequences of their interactions in CD, cell wall integrity and pathogen proliferation are unknown. It is known that the MtrAB two-component signal transduction system and the PknA/B serine-threonine phosphorylation kinases signaling system are two of the many regulatory signaling networks that Mtb uses for its optimal survival. Our findings also suggest that MtrB plays important roles in CD/CWS, interacts with PknA/B and the interaction partners of CrgA and CwsA. However, the regulatory roles that MtrB plays in Mtb CD/CWS and pathogen proliferation are unknown. This proposal focuses on the CD process mediated by CrgA and CwsA proteins and its regulation by the MtrAB and PknA/B signaling networks. Specific Aim 1 investigates a hypothesis that interactions of Mtb CrgA and CwsA with each other and their partners play vital roles in the CD/CWS; defects in their activities and interactions compromise CD/CWS, hence pathogen proliferation. Specific Aim 2 investigates a hypothesis that MtrB performs two regulatory roles: (i) modulates the expression levels of the MtrA-target genes engaged in CD/CWS and pathogen proliferation, (ii) modulates the PknA/B target phosphorylation critical for CD/CWS, and that MtrB interactions with its partners dictate the outcome of these activities. We will investigate both roles by creating and characterizing MtrB-depleted strains and mtrB mutants defective for select pair of interactions under growth conditions relevant for Mtb life style. Our studies will provide a deeper understanding of the Mtb multiplication mechanisms upon infection, hence will lead to anti-TB drug discovery for preventing TB related mortalities.